not degrade host DNA containing the recognition sequences and they must only cleave DNA molecules that contain recognition (cognate DNA) sites without cleaving DNA molecules that lack these sites

·Restriction enzymes must cleave DNA only at __, without cleaving at other sites

oTo keep from damaging host-cell DNA, restriction enzymes must cleave __ much more than 4000 times as efficiently as they cleave nonspecific sites

·Cleavage is by __of __ from __by __

oA __ catalyzes the __

§Specifically, the bond between the 3’-oxygen atom and the phosphorus atom is broken

recognition sites

cognate DNA molecules

in-line displacement

3’-oxygen

phosphorus

magnesium-activated water

restriction endonuclease

hydrolysis of the phosphodiester backbone of DNA

oThe products are DNA strands with a free 3__ and a __ at the cleavage site

§This reaction proceeds by __ at the phosphorus atom

·Two mechanisms are used:

oMechanism one: __

oMechanism two: __

’-hyroxyl group

5’-phosphoryl group

nucleophilic attack

through a covalent intermediate, employing a potent nucleophile

direct hydrolysis

·In either case, each reaction takes place by __. Explain it in depth.

in-line displacement

The incoming nucleophile attacks the phosphorus atom, and a pentacoordinate transition state is formed, which has trigonal bipyramidal geometry centered at the phosphorus atom, with the incoming nucleophile at one apex of the two pyramids and the group that is displaced at the other apexà inversion

·The two mechanisms differ in the __

number of times that the displacement takes place int eh cource of the reaction

oFirst type: __

§It is then __to produce the final products; and, ultimately, __ take place at the phosphorus atomà __--> no change in __

a nucleophile in the enzyme attacks the phosphate group to form a covalent intermediate

hydrolyzed

two displacement reactions

double inversion

stereochemistry

oSecond type: __

§A __ takes place at the phosphorus atomà __

an activated water molecule attacks the phosphorus atom directly

single displacement reaction

inversion

How do they determine which type it is? What was revealed?

·oxygen is replaced with sulfur and water that is coming in is enriched in 18O to allow the incoming oxygen atom to be marked. The location determines whether it is inversion or retention.

oThe analysis revealed that the stereochemical configuration at the phosphorus atom was inverted only once with cleavage

oIn one, crystals of EcoRV endonuclease are prepared bound to __that contain the __and grown in __. They are then soaked up. They can also be grown in a __; or, they can be __

§In all cases, no cleavage takes place

magnesium

oligobucleotides

enzyme’s recognition sequence

absence of magnesium

mutated enzyme

replaced with other nonmetallic ions that don’t result in much catalytic activity

·One ion binding site is occupied in all structures; and, the metal ion is coordinated to the protein through __ and __ near the __. This metal ion binds the __ that __, helping to __ and __ it in a manner similar to that for the Zn2+ ion of carbonic anhydrase

·The recognition sequences for most restriction enzyme are __, which gives the __

two aspartate residues

to one phosphate group oxygen atoms

near the site of cleavage

water molecule

attacks the phosphorus atom

position and activate

inverted repeats

3D structure a twofold rotational symmetry

Structure of enzymes

oDimers whose two subunits are related by twofold rotational symmetry, which facilitates the recognition of cognate DNA by the enzyme

·An enzyme’s __ for substrates often determines __. The specificity doesn’t come in the __, but the __. The answer lies in a unique set of interactions between the enzyme and a cognate DNA sequence

oWithin the 5’-GATATC-3’ sequence, the G and A bases at the 5’ end of each strand and their Watson-Crick partners directly do what?

binding affinity

specificity

type of DNA bound

sequence that gets cleaved

contact the enzyme by hydrogen bonding with residues that are located in two loops, one projecting from the surface of each enzyme subunit

§The most striking feature of this complex is the __, which is __in the center. The central two __ play a key role in producing the __. They do not make contact with the enzyme but appear to be required because of their __

·5’-TA-3’ sequences are known to be among the most easily __. The distortion of the DNA at this site has severe effects on the __

distortion of the DNA

kinked

TA base pairs in the recognition sequence

kink

ease of distortion

deformed base pairs

specificity of enzyme action

oSpecificity can be determined by __

§Although EcoRV endonuclease (for example) binds to both __ and __ with equal affinity, the structures of complexes formed are different:

·The noncognate DNA conformation is not __--> no __ is positioned sufficiently close to the __ to __; and, the nonspecific complexes don’t __ and the __. The distortion of substrate and subsequent __ account for the __ of more than 1,000,000-fold that is observed for EcoRV endonuclease despite little preference at the level of substrate binding

enzyme’s binding affinity for substrates

cognate and noncognate DNA

distortedà

phosphate

active-site aspartate residues

complete a magnesium ion binding site

bind the magnesium ion

catalytic apparatus is not assembled

binding of the magnesium ion

catalytic specificity

§Thus, __ is needed to attain __. When binding to enzymes, the DNA is distorted in such a way that __, increasing the __.

binding energy

catalytic specificity

additional contacts are made between the enzyme and substrate

binding energy

But, this increase is canceled by the __. Thus, for EcoRV endonuclease, there is little difference in __ for cognate and nonspecific DNA fragments. However, the distortion in the cognate complex dramatically affects catalysis by completing the __

energetic cost of distorting the DNA from its relaxed conformation

binding affinity

magnesium ion binding site

oThis illustrates how enzymes can utilize available __ to __ and poise them for __. Interactions that take place within the distorted substrate complex __, leading to __

binding energy

deform substrates

chemical transformation

stabilize the transition state

DNA hydrolysis

§Distortion in the DNA explains how __blocks catalysis and protects host-cell DNA. Explain.

methylation

When a methyl group is added to the amino group of the adenine nucleotide at the 5' end of the recognition sequence by enzymes called methylases, the methyl group's presence precludes the formation of a hydroen bond between the amio group and the side chain carbonyl group of the asparagine, which is closely linked to the other amino acids that form specfic contacts with the DNA. The absence of the hydrogen bond disrupts other interactions between the enzyme and the DNA substrate, and the distortion necessary for cleavage will not take place

·An endonuclease will not cleave DNA if its __ is __. These pairs of enzymes are __

oThe distortion in the DNA explains how methylation does what?

recognition sequence is methylated

restriction-modification systems

blocks catalysis and protects host-cell DNA.

·Restriction enzymes are not significantly similar. But, the 3D structures revealed the presence of a core structure conserved in the different enzymes, which includes __--> evolutionarily related

o__ may have led bacteria to acquire the similar genes; so, they may have obtained the gene for these restriction endonucleases from a common source more recently than the time of their __